Bacteriostat



2,904,462 Patented Sept. 15,, 1959 'BACTERIOSTAT Marjorie E. Moreland, Summit, and Daniel H. Reheis, Maplewood, NJ.

No Drawing. Application May 3, 1956 Serial No. 582,321

1 Claim. (Cl. 167-14) This invention relates to bacteriostasis.

The 'bacteriostatic agent is an aluminumchlorhydroxy complex of the type formula A1,,(OH) ,01,, in which x is an integer and y and z are-numbers whose sum is 3x.

A particularly satisfactory product'for the present purpose has the empirical formula Al (OH) Cl.

Such material has been used heretofore as an astringent. We have now discovered that'the complex, when used in aqueous solutions so dilute as to be-inefiective as astringents, is effective as a bactericide or bacteriostat. I

Briefly stated, the invention comprises an agentin the form of very dilute aqueous solutions of an aluminum chlorhydroxy complex for inhibiting the growth of microorganisms and also the process of contacting the microorganisms with the complex of such dilutions. In the most satisfactory embodiment, the invention comprises the complex as a bacteriostat at dilutions corresponding to approximately l0 -2.5 10 parts of water for 1 of the aluminum chlorhydroxy complex onthe dry basis. At higher concentrations, the complex kills much smaller numbers of bacilli per unit of complex used.

We have found a peak in'the effectiveness atsuch extreme dilutions. Tests were made, for example, as follows: To each of five dilutions of aluminum chlorhydroxy complex 1 ml. of the micrococci bacterial suspension containing a known number of organisms, ordinarily from 500,0001,200,000 per ml. was mixed with the said complex at room temperature and allowed to stand for selected periods of time. 1 ml. was then transferred from each dilution to a petri dish and 10 ml. Difco 'Bacto- Tryptose agar was added. Plates were allowed-to solidify and then placed in a 37 C. incubator for 48 hours. The petri dish was then examined with a Quebec colony counter and the number of colonies determined in accordancewith usual technique, the number of colonies counted showing the number of bacteria remaining viable 50 after contact with the complex. The counts given herein are for the most part averages of two or more tests for each dilution of the complex.

Parts of water, pH of solution approx, for 1 part Number of coloniesafter of dry aluminum incubation chlorhydroxy Complex Complex complex 1 +br0th 1 The number of parts of water used were actually about of those shown-in all dilutions stated here and elsewhere herein. Thus the figure 10,000 stated exactly should be 9,000. The figures are rounded as shown, however, to avoid the impression of an accuracy of degree'that-does not prevaikin bacteriological work of this kind.

and to use.

The complex used in obtaining the data tabulated above contained 1.5 Al for 1 O1.

Proportions are expressed as parts by Weight in all cases unless specifically stated to thecontrary.

As to materials used, the aluminum chlorhydroxy complex used is a commerical grade of high purity such as made by the interaction of aluminum powder with "aluminum chloride, as descn'bed is Patent 2,196,016, is-

sued to Huehn et al., on April 2, 1940. The aluminum powder and the chloride are proportioned to give the ratio A1:Cl desired in the finished complex.

When the proportion of aluminum in the complex is less than 1.5 atoms for 1 of chlorine, there is a decided loss of bacteriostatic effect. When the proportion is more than 2.5 :1, the complex 'is diflicul-t to make The aluminum chlorhydroxy complex at high concentrations, as above 1 part for of water has a tendency to precipitate proteins, as shown by its effect on the agar composition used for test purposes by us.

The complex improves in effectiveness with age, as from 2 weeks to 2 months, the effect of age as Well as promoters being illustrated 'in Example 3. I r

The bacteria to be contacted with the solution of the complex may be either gram positive, such Micrococci, or gram negative, such as Escherichia and Aerobacter.

The water used in all tests reported herein was soft (substantially free from the ions of hard water, i.e. calcium, magnesium, sodium, sulfate, chloride, and bicarbonate ions) and, more specifically, was distilled water unless specifically stated to the contrary. The pH of freshly made solutions of the chlorhydroxy complex, at all dilutions up to the 1:10 ran below 6.5.

Cultures held 3, 9, and 14 minutes in contact with the complex at high dilution before mixing with the agar in a petri -dish showed that the bacteriostasis increases with time. The major part of the effect, however, comes in the first 3 minutes. In the test tabulated above, for instance, the results for various periods with the dilution 10 parts of water for 1 of the complex, for various periods of contact before incubation and with an original population of 1,000,000 organisms per ml., areas follows:

Remaining viable bac- Mmutes after teria as shown by inoculation colonies after incuwith micrococci bation at 38 C. for

The exact temperature'of mixing the bacterial suspen-- sion and the chlorhydroxy complex is relatively unimportant. In tests of mixing at 28, 35, and 45 C., the results after incubation have been found to be approximately the same.

The invention will be further illustrated by description in connection with the following specific examples of the practice of it.

EXAMPLE 1 An aluminum chlorhydroxy complex containing 2 atoms of aluminum for each atom of chlorine and containing iron in proportion less than 50 parts for 21 milto an atomic ratio of ca. 1.5 A1 for 1 Cl.

lion of the complex, is dissolved in water in amount to correspond to 20 parts on the dry basis for 80 of water.

The solution so made is ready for dilution and subsequent use as a bacteriostat.

EXAMPLE 2 EXAMPLE 3 This example illustrates the use of promoter additives with the aluminum chlorhydroxy complex.

Glycerine is particularly effective as the additive. Other non-toxic polyhydric alcohols that illustrate the class of satisfactory materials are propylene glycol and sorbitol.

Another class of additives that are satisfactory are non-toxic surface active agents. Examples are the nonionics such as polyalkylene oxide derivatives of the partial esters of sorbitan, sorbitol, and glycerine with the higher C C fatty acids such as lauric, oleic, and stearic. The number of alkylene oxide groups introduced into the partial ester (monoor di-) may vary over a considerable range, as for instance from 1-100 and is suitably -25. The alkylene oxide ordinarily is ethylene oxide, although the propylene and isopropylene oxides may be used. Commercial products of this kind that we may use are those known as Span and as Tween. Other surface active agents that may be used are glycerine and ethylene glycol, both in the form of monoesters with the higher fatty acids listed above.

The chlorhydroxy complex containing the additives were tested for bacteriostatic effect With essentially the same technique as described earlier herein.

In these particular tests, the mixture of the bacterial suspension with the chlorhydroxy complex, additive, and water was held for 3 minutes after mixing before the agar was introduced. The incubation that followed was at 37 C. for a period of 48 hours.

Most of the numerical counts of colonies tabulated below are averages of several runs made on different days.

The counts of the colonies after incubation were compared, for each additive and complex, with a control in which the chlorhydroxy complex was omitted.

The proportion of the additive was 25 parts by weight in every instance for 100 of the aluminum chlorhydroxy complex on the dry basis.

In all cases, the aluminum chlorhydrox-y complex contained aluminum in the proportion of 2 atoms for 1 atom of chlorine. The complex was of two grades, referred to below as regular and special, respectively. The

regular was freshly made by reaction of aluminum powder and aluminum chloride, as described earlier herein, and was filtered. The special was made similarly except that the filtrate (containing the said complex) was vacuum dried to a content of 47% of aluminum calculated as A1 0 The dried material was then dissolved promptly in water and kept in solution at ambient temperatures for 68 days before use in the test with glycerine additive and for 77 days before test with the additive Tween.

The tables that follow show the dilutions at which the bacteriostatic compositions were tested and the count of colonies after incubation. In some of these runs there was a variation in the bacterial count from a finite number to numbers too numerous to count (TNTC). When this variation was found, it is so indicated in the table.

Glycerine plus chlorhydroxy complex Count after incubation Parts of water for 1 part; a

of mixed glycerine and Control, complex, dry basis Regular Special glycerine complex complex with no complex TNTO 1866TNTG TNIO It will be seen, by comparing the data above with that earlier herein, that the glycerine additive is a promoter of the bactericidal or bacteriostatic effect.

The additive used in obtaining the following data was polyethylene oxide derivative of sorbitan monolaurate (Tween).

Various proportions of the additive promoter may be used as for instance 1%-10% of the weight of the aluminum chlorhydroxy complex. Proportions substantially below 1% are ineffective and those above 10% are unnecessary, and, therefore, uneconomical.

The tests of this Example 3 show that the aged (special) complex was more effective than the regular.

Another promoter that may be and suitably is admixed with the aluminum chlorhydroxy complex is an alkali metal iodate such as sodium or potassium iodate. Thus, We have used potassium and sodium iodates. The proportion recommended is 2-15 parts for parts of aluminum chlorhydroxy complex on the dry basis, best results having been obtained when the proportion of the iodate is not above 5- parts of the iodate for 100 parts of the complex.

A feature of the use of iodates with the complex is the killing at very great dilutions. In a representative run, using 5 parts of the potassium iodate for 100 of the aluminum chlorhydroxy complex and the techniques described earlier herein, we have found the following counts of Micrococci for the various dilutions of the complex andiodate mixture:

Parts of water for 1 of aluminum hydroxy complex Count attrmixed with 0.05 part of K10 incubation 10 TNIC 10 123 10 15 10 0 10 800 The results, here as elsewhere, vary somewhat in work minutes, and 14 minutes with all dilutions from 100 parts of water to 1,000,000.

EXAMPLE 4 Other additives that may be mixed with aluminum chlorhydroxy complex before contact with the bacteria are the following: Phospholipids of which the lecithins, cephalins, and sphingomyelins are examples, reducing agents such as soluble bisulfites and dithionates of which the sodium salts are satisfactory and the most economical; oxidizing agents of which examples other than the iodates are the chromates and manganates such as sodium or potassium chromate and potassium permanganate; and surface active agents other than those described above, such as anionics including the sodium or potassium alkyl aryl sulfonates, lauryl sulfonate, and alkyl sulfates.

EXAMPLE 5 Count after incubation Parts of water for 1 part of aluminum chlorhydroxy complex Distilled water Hard water TNTO 0 While various explanations may be advanced to explain these results with the hard water, it is considered that an important part of the eifect may be the decomposition or change of structure of the aluminum chlorhydroxy complex by ions in the hard water, the ion eontent as a whole causing the pH to be above 7 and consisting principally of calcium, magnesium, sodium bicarbonate, sulfate, and chloride.

The hard water used in the tests of this example was the city water supply of Summit, New Jersey, of pH about 7.25 and the following approximate analysis, the Ca, Mg, Cl, and Na being reported as the elements.

Determination: P.p.m. Alkalinity, total 106 Hardness, permanent 32 Hardness, total 138 Carbonic acid, free 2.6

6 Total solids 225 Iron and aluminum, calc. as oxides 0.7 Calcium 29.9 Magnesium 7.9 Chloride 9.0 Sodium 14.9 Nitrites .001 Nitrates 0.7

EXAMPLE 6 The procedure of Examples 1-3 is followed except that the fungi Trichophyton interdigitales was the organism to which the dilute solution of the complex was applied.

EXAMPLE 7 The procedure of Examples 1 or 3 is followed except that the proportion of Water is decreased to IO Xthe amount of the aluminum chlorhydroxy complex on the dry weight basis. With the distilled or soft water, even higher concentrations, as up to 1 part of the complex to 5 parts of water, are satisfactory but unnecessary.

It is to be understood that it is intended to cover all changes and modifications of the examples of the in-- vention herein chosen for the purpose of illustration which do not constitute departures from the spirit and scope of the invention. It is to be understood also that the aluminum chlorhydroxy complex is not only a bacteriostat but also a bactericide.

What is claimed is:

The process of inhibiting the growth of bacteria which comprises applying to the bacteria and maintaining in contact therewith for a period of at least three minutes a bacteriostat consisting essentially of an aqueous solution of aluminum chlorhydroxy complex of the empirical type formula Al (OH) Cl,, in which x is a number within the range about 1.5-2.5, z is 1, and the sum of y and z is 3x, the solution being in the condition of having been aged at ambient temperatures for at least 2 weeks and the concentration of the said complex at the time of application to the bacteria being about 1 part for 10 to 2.5 x 10 parts of water.

References Cited in the file of this patent UNITED STATES PATENTS Andersen Dec. 20, 1949 OTHER REFERENCES 

